DNA polymerases synthesize the formation of DNA molecules from deoxynucleoside triphosphates using a complementary template DNA strand and a primer. DNA polymerases synthesize DNA in the 5'-to-3' direction by successively adding nucleotides to the free 3'-hydroxyl group of the growing strand. The template strand determines the order of addition of nucleotides via Watson-Crick base pairing. In cells, DNA polymerases are involved in repair synthesis and DNA replication.
Bacteriophage T5 induces the synthesis of its own DNA polymerase upon infection of its host, Escherichia coli. The T5 DNA polymerase (T5-DNAP) was purified to homogeneity by Fujimura RK & Roop BC, J. Biol. Chem. 25:2168-2175 (1976). T5-DNAP is a single polypeptide of molecular weight of about 96 kilodaltons. This polymerase is highly processive and, unlike T7 DNA polymerase, does not require thioredoxin for its processivity (Das SK & Fujimura RK, J. Biol. Chem. 252:8700-8707 (1977); Das SK & Fujimura RK, J. Biol. Chem. 254:1227-1237 (1979)).
Fujimura RK et al. J. Virol. 53:495-500 (1985) disclosed the approximate location of the T5-DNAP gene on the physical restriction enzyme map generated by Rhoades (J. Virol. 43:566-573 (1982)). DNA sequencing of the fragments of this corresponding region was disclosed by Leavitt MC & Ito J, Proc. Natl. Acad. Sci. USA 86:4465-4469 (1989). However, the authors did not reassemble the sequenced fragments to obtain expression of the polymerase.
Oligonucleotide-directed, site-specific mutation of a T7 DNA polymerase gene was disclosed by Tabor S & Richardson CC, J. Biol. Chem. 264:6447-6458 (1989). It is believed that there are no publications before the invention of the subject matter disclosed herein describing mutation of 3'-to-5' exonuclease activity of T5-DNAP.
The existence of a conserved 3'-to-5' exonuclease active site present in a number of DNA polymerases was predicted by Bernd A et al., Cell 59:219-228 (1989).
In molecular biology, DNA polymerases have several uses. In cloning and gene expression experiments, DNA polymerases are used to synthesize the second strand of a single-stranded circular DNA annealed to an oligonucleotide primer containing a mutated nucleotide sequence. DNA polymerases have also been used for DNA sequencing. Tabor & Richardson, U.S. Pat. No. 4,795,699, disclose that processive, host thioredoxin-requiring, T7-type DNA polymerases, especially those lacking substantial exonuclease activity, are very useful for DNA sequencing.